Project Summary/Abstract The human cerebral cortex consists of billions of cells that are primarily generated during developmental stages. During neural development, the neuroepithelium gives rise to radial glia, which is the canonical neural stem cell. Radial glia then asymmetrically divide into transit amplifying intermediate progenitors, which differentiate into excitatory neurons. These steps of neurogenesis have been well characterized in the literature. However, there have been very few studies dedicated to understanding the molecular identity of neuroepithelial stem cells, exploring the transition from neuroepithelial stem cell to radial glia, and teasing apart their contribution to the neocortex. Here I leverage single-cell RNA sequencing data from primary cortical samples through the BRAIN Initiative to reveal evidence of heterogeneity of neural stem cells in the first trimester. Several gene candidates have already been identified through my analyses that are enriched in progenitors during early first trimester development. I have identified two genes, DLK1 and HES4, both of which are interestingly non-canonical players in the Notch signaling pathway. DLK1 and HES4 are both enriched immediately in different progenitor populations before the switch from neuroepithelial stem cell to radial glia. Lineage trajectory analysis using RNA velocity demonstrates a clear putative trajectory from the neuroepithelial stem cell clusters to radial glial clusters. When I enriched for genes that influence RNA velocity the most, DLK1 and HES4 were among the top genes influencing the cell fate switch. Therefore, I hypothesize that DLK1 and HES4 are markers for neuroepithelial stem cells, and are important during the transition from neuroepithelial stem cell to radial glia. To test my hypothesis, I will be performing genetic modulations of DLK1 and HES4 in cerebral organoids to determine if these genes are necessary and/or sufficient in neuroepithelial stem cell production.